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Immunological and non-immunological synthesis and release of prostaglandins and thromboxanes from isolated guinea pig trachea
PROSTAGLANDINS
IMMUNOLOGICAL AND NON-IMMUNOLOGICAL SYNTHESIS AND RELEASE OF PROSTAGLANDINS AND THROMBOXANES FROM ISOLATED GUINEA PIG TRACHEA. J. F. Burka, M. Ali, J. W. D. McDonald and N. A. M. Paterson Department of Medicine, University of Western Ontario, London, Ontario, Canada. N6A 4G5 ABSTRACT Specific radioimmunoassays were used to demonstrate the synthesis by the guinea pig trachea of 6-keto PGFlu, TxB2, and PGF2o in addition to PGE2. The rank order of both spontaneous and stimulated release was PGE2 > PGF > 6-keto PGFlo = TxB2. Ovalbumin-induced prostanoid release $" rom sensitized tissue was antigenspecific. The release was unlikely to be a secondary consequence of tracheal contraction since incubations with calcium ionophore A23187, at a concentration which produces an equivalent magnitude of contraction of sensitized trachea, did not induce a significant PG or TX production. In contrast, significantly higher prostanoid synthesis was induced by A23187 in unsensitized than sensitized trachea. Thus sensitization altered the profile of arachidonic acid metabolism evoked by the ionophore. Correspondence: J. F. Burka, Department of Pharmacology, University of Alberta, 9-70 Basic Medical Sciences Building, Edmonton, Alberta, Canada. T6G 2H7 INTRODUCTION Activation of arachidonic acid (AA) metabolism occurs following antigen challenge of perfused sensitized guinea pig lung (1) or sensitized tracheal strips (2, 3). The major cyclooxygenase products released immunologically from isolated perfused guinea pig lung are prostaglandin (PG)F2e, prostacyclin (PGI2), thromboxane (Tx)A2 and their metabolites (4). In the guinea pig both large and small airways contract in response to antigen challenge. Since the guinea pig trachea reacts similarly to human bronchus to histamine and slow-reacting substance of anaphylaxis (SRS-A) (5), we have used this model to examine the airways contraction in response to immunological and non-immunological stimuli (2, 3). Indomethacin, a cyclooxygenase inhibitor, reduces airway tone and enhances the contraction induced by antigen, histamine, carbachol (2, 6) and calcium ionophorc 1123187 (7). This may in part be due to inhibition of PG synthesis by the trachea. To date PG release from trachea has been measured only by superfusion bioassay, with which a PGE-like material has been detected (2, 3, 8, 9). We now report the use of specific radioimmunoassays to demonstrate that PGF2o, PG12 and TxA2, in addition to PGE2 are released from the trachea by both immunologic and non-immunologic stimuli.
NOVEMBER
1981 VOL. 22 NO. 5
683
PROSTAGLANDINS
Tracheae were removed from anesthetized male English shorthair guinea pigs (200-300 g) (Connaught Laboratories, Toronto, Ontario), cleaned of extraneous tissue, spirally cut (lo), divided into three equal segments and well washed in Krebs-Henseleit solution. Sensitized tracheae were obtained from guinea pigs immunized 2-4 weeks previously with ovalbumin (DA) 100 mg. S.C. and 100 mg i.p. The first segment was added immediately to 30 ml 07% ethanol. The remaining tracheal tissue was placed in 10 ml oxygenated KrebsHenseleit solution and pre-incubated at 37'C in a gently shaking water bath for 5 minutes. The second segment was incubated for a further 30 min in Krehs solution (i.e. no stimulus added). The third was incubated for 30 min in Krebs solution to which was added AA (100 uM), CIA (10 pg/ml), or A23187 (5.7 yM). The reaction was stop ed by the addition of 2 volumes absolute ethanol, with 0.03 UCi !_ H PGF2 added for recovery analysis. The pH was adjusted to 3 with 88% formic acid and 2 volumes chloroform added. The mixture was well shaken, centrifuged to facilitate phase separation and the chloroform phase evaporated under reduced pressure to approximately 1 ml. The remainder of the solvent was evaporated under N2, the residue resuspended in 0.5 ml 2% methan~\l : 98% chloroform and applied to an activated silicic acid column. 'The column was then eluted successively with 8 ml of 2% methanol in chloroform which removes unused precursor and relatively non-polar products (ll), followed by 8 ml of 10% methanol in chloroform (which contained the more polar prostaglandins and thromhoxanes). The eluate was evaporated under N2 and the residue resuspended in distilled absolute ethanol. Radioimmunoassays of PGE2, PC,F2,,TxB2, and k-keto PGFlcrwere performed as described previously (12). Differences in the amount (rig/gwet weight) of prostnglandin and thromboxane release by trachea in the absence or presence of stimuli were analyzed for significance by the use of Student's t-test (one-tailed) for paired data (13). Comparisons of release from sensitized and unsensitized tissue were analyzed for significance by multivariate analysis using Hotelling's T2 test (2-tailed) (14). OA- and A23187-induced release of prostanoids from sensitized tissue were also compared using Hotelling's T2 test. MATERIALS. Ovalbumin (Grade II for sensitization and Grade III for challenge) and arachidonic acid (Grade I) were purchased from Sigma; calcium ionophore A23187 from Calhiochem; and 3H-PGF2, from Amersham. We are grateful for the gifts of prostaglandins and thromboxane B2 from Upjohn and indomethacin from Merck-Frosst. Indomethacin was dissolved in 1 11Tris buffer (pll8.4), diluted in saline, and kept on ice. AA was stored as a 10 m$;/ml stock solution in hexane under
684
NOVEMBER
1981 VOL. 22 NO. 5
PROSTAGLANDINS
A Tris salt was prcpnred with 1 M Tris buffer (pR 8.4) N’. centration of 1 mg/mi, dilrltcd in sal ine and kept on ice.
to a con-
The chloroform and methanol lrsed in the extraction process were Absolute ethanol was redistilled. toxigrnphic: grade (Annchemia). All other solvents were reagent grade. ~hc silicic acid column was prepared with Bio-Sil A (200-400 A slurry of silicic mesh) (biorad) activated at 115OC for 1 hour. acid in 2L methnnol in chloroform (1:4 wt:vol) was prepared and The column approximately 4 ml added to each column (0.8 x IO cm). in chloroform W;IS waslretl with 6 ml of enc,h of 20% and 2% methanol before application of the snmple. RESULTS. ---~In pilot experiments it was found that maximal release of prostanoids from trachea requirecl 30 min incubation with 10 ug/ml These conditions were OA (in sensitized tissue) or 100 LIM AA. The concentration therefore employed for the remainder of the study. of A.23187 used (5.7 LIM) was that which produced the same magnitude of tracheal contraction as OA (10 pfi/ml) (7). how amounts of h-keto PGFL,r and TxR7L as well as higher amounts of PGE2 and P(:P2o were detected ni~ter immediate extraction of both incubation of the trachea for sensitized and unsensitized trachea. In all situations, 30 min in buffer alone led to additional PG release. including specific stimulatjon witlr AA, CA, or A23187, the rank order of prnstano-itls released was KC2 -- PGF2,> > h-keto P!:FLixg TxR2. iJhen exoj:enous AA (100 tIPI), the natural substrate for cyclowas added to the trachea there was a significant (p d 0.05) oxvgenase increase in the reLease of all four cyclooxygcnase products in both sensitized and unsensitized tracheae.
In sensitized trachea, CIA (10 pg/ml) induced significant increments in tire production of 0-keto PGFLIL, PGE2, and PGF2, (Table The rise in T::B2 approached significance at tile p < 0.05 level Hultivurinte .~nnlysis <.onfirmed a highly significant (p ': 0.07). djfference (p I' 0.001) between the effects of OA on sensitized and In contrast, bovine serum
1).
The calcium ionophore A23187 (5.7 ;IM) failed to stimulate additional PC: or TX relcasc from sensitized trachea (Table 1). In unsensitized tissire nlthou~:l~ the release of the individual prostanoids
NOVEMBER
1981 VOL. 22 NO. 5
685
3
ti
e
4
4
(5.7 PM)
A23187 0
0
(5.7 PM)
OA (10 &ml)
0
0 AA (100 PM)
0
0 0 A23187
87.9 41.3 I 4.6 17.5
14.4 T f 4.8
87.5 54.1 + 5.0 21.4
11.4 T + 1.8
53.2 + 7.3 38.4 + 3.7
27.6 + 2.3 63.3 2 5.5 153.6 + 12.2****
7.6 + 2.0 22.8 + 5.1 99.0 z 21.5* 47.1 f 11.4 47.3 z 7.7
4.6 + 0.7 38.9+ 5.5 34.8 z 0.4
24.0 + 4.0 63.0 + 12.6 172.8 + 21.0***
38.7~+ 4.6 124.6-L 6.5****
4.6 + 0.3
6-keto PGFla
4.1 + 0.6 40.7-+ 16.1 54.0 + 11.4
15.4 + 7.2 101.0+ 8.7 251.9 7 - 75.2
26.0+ 10.1 119.9~+ _ 14.1***
3.2 + 1.3
TxB2a)
462.2 371.6 T + 60.8 110.7
289.9 f 56.4
250.9 t 26.3 334.8 + 53.2
373.3 + 72.9 493.2 + 68.0** 962.4 + 89.3
144.3 + 12.5 251.1 T 33.7 193.1 z 16.6
570.0 + 117.5 899.6 + 79.9 1225.4 -+ 149.4*
208.1 ? 24.8 988.4 z 22.3****
110.6 + 11.4
PGE2
76.3 101.4~+ +- 19.7 30.3
30.2 + 9.7
107.9 + 10.0 86.1 k-4.6
82.7 + 7.1 141.9--t 1.5 398.5 + 25.1**
12.5 + 1.2 46.3 T 5.1 44.9 + 1.9
72.2 •t 7.2 151.8+ 27.0 384.8 T - 20.8****
51.2 + 4.3 211.3+ - 16.4**
16.3 + 0.5
PGF2a
trachea
Statistical significance (* p < 0.05; ** p < 0.025; *** p < 0.01; **** p < 0.005) Values are mean + S.E.M. calculated by Student's t-test for paired data comparing values at 30 min with and without stimulus.
a)
30
2 6 3
4
0 30 30
2
4
0 30 30
.:I? 0
4
4
0 AA (100 UM)
0 0 OA (10 ug/ml)
0
Stimulus
30 30
n
Production of TxB2, 6-keto PGFl,, and PGF2U (rig/g wet weight) by sensitized and unsensitized following stimulation with ovalbumin (OA), AZ.3187 or arachidonic acid (AA).
0
Incubation Time (min)
Table 1.
5; 00
E
5
0
PROSTAGLANDINS
did not achieve significance at the p < 0.05 level, multivariate analysis indicated total prostnnoid synthesis was significantly (p < 0.008) higher than in sensitized trachea.
The present study demonstrates that PGE2 is the major cyclooxygenase product synthesized in guinea pig trachea both spontaneously and following stimulation with M or CA. The pattern is in contrast to guinea pig perfused lung and parenchymal preparations where 6-keto PGF1, and TxB2 are the major AA products (15, 16). This study provides the first evidence that cyclooxygenase products in addition to PGE2 are generated by the guinea pig trachea. Previously, we (2) and others (9) using biological assay methods (i.e. cascade superfusion) detected only a PGE-like substance released by guinea pig trachea in response to CA, AA, ncetylcholine, and histamine, although Orehek et al. (8) reported the release of PGF2c(. Although superfusion bioassay is invalrlahlefor discriminating simultaneously between various M metabolites and for detection of unstable intermediates, the method is limited by its lack of sensitivity. Whereas the threshold for PG12-induced relaxation of the bovine coronary artery is 20-100 ng/ml (17), concentrations as low as 70 pg/ml 6-keto PCFItx (a stable hydrolysis product of PCT2) can be detected by radioimmunoassay. Similar high sensitivities for TxB2, PGE2, and PGF2a are obtainable with radioimmunoassay. Thus it is not surprising that PGI2 and thromboxanes released from the guinea pig trachea were not detected in studies using biological assay methods. The rank order of prostanoids released was PGE2 3 PGF2a > 6-keto PGFle e TxB2. AA normally relaxes the guinea pig trachea (2), an observation which is consistent with the present observation that PGE2 is the major cyclooxygenase product of M metabolism by the trachea. M metabolitcs are not stored to any considerable extent and any increase in their levels induced by a physiological or pathological stimulus occurs through their rapid biosynthesis. Thus it is likely that the basal levels of PGs and Txs detected in these experiments are a consequence of tissue dissection and preparation. Furthermore, the spontaneous generation of PGs and Txs during the 30 min incubation period may have resulted at least partly from membrane pertubations induced by the gentle agitation. Mild mechanical stimulation has previously been shown to induce PG release (19). On the other hand k~ontinuousproductjon of prostanoids in gastrointestinal and tracheal smooth muscle has been reported to contribute to resting tone (8, 18) and the observed spontaneous release may thus reflect physiologic turnover. OA contracts sensitized guinea pig trachea (2) and induces the release of PGE2 and F2d and smaller amounts of 6-keto PGFJ, and TxB2. OA-induced prostanoid release is a consequence of an antigen-specific immunological reaction on sensitized tissue. However it is not known whether M metabolism is activated directly by the immunological
NOVEMBER
1981 VOL. 22 NO. 5
687
PROSTAGLANDINS reactions, as has been demonstrated in rat basophilic leukemia cells (20) or secondarily by activation of phospholipase by other released mediators, such as histamine and SRS-A (3, 9, 21). Tt is also possible that the PG release may be secondary to tracheal contraction (8, 9). In the present experiments A23187, employed at a concentration which produces an equivalent magnitude of tracheal contraction (7) did not induce significant PG or TX production. Thus qmthesis of the cyclooqgenase products PGE2, PGF2o, PG12, and TxA2 by trachea is not an inevitable concomitant of smooth muscle contraction. Diegel et al. (22) have suggested that separate calcium pools are involved in smooth muscle contraction (guinea pig taenia coli) and PG synthesis and that the two processes can be dissociated. Whereas 012 initiates an immunological reaction leading to a series of intracellular biochemical events including entry of calcium into the cell (20, 23), calcium ionophore A.23187bypasses the membrane associated events (24) and can activate phospholipase AT by direct introduction of cnlrium into cells (25). A23157 has bc;n reported to stimulate I'(: and TX biosyntllesis in platelets (25, 26) and mast cells (27). It is interesting that significantly higher prostanoid production was induced hy A23167 in unsensitized than sensitized trachea. Sun et al. (28) have suggested that the kinetics of the cyclooxygcnnse differ between sensitized and unsensitized tissue. A fast cyclooxygenase in sensitized tissue would provide lligher amounts of PGR2 which would favour TX synthesis. However, this was not seen in trachea in our experiments and other explanations must be sought. It is possible that cyclooxygenase products other than those measured in our experiments are produced in response to A23187 or that A23187 preferentially stimulates the lipoxygenase pathway of AA metabolism. Both A23187-induced contraction and the prolonged phase of OA-induced contraction are reduced with the cyclooxygenase/ lipoxygenase inhibitor, phenidone, providing indirect evidence that lipoxygenase products contribute to OA- and A23187-induced contractions (2, 7). ACKNOWLEDGENENTS. This project was supported by the Medical Research Council of Canada, the Ontario plinistryof Health, and the Ontario Heart Foundation. We thank Mr. Warren YcDonald and Mr. Chris Webb for their technical assistance, Dr. George Wells for statistical analysis, and Mrs. Sharon Hughes for typing the manuscript.
688
NOVEMBER
1981 VOL. 22 NO. 5
PROSTAGLANDINS
RliFEfENCES. ---1.
Piper, P. J. and J. R. Vane. Release of additional factors in anaphylaxis and its antagonism by anti-inflammatory drugs. Nature 223: 29, 1969.
2.
Burka, J. F. and N. A. M. Paterson. Evidence for lipoxygenase Prostaglandins involvement in allergic tracheal contraction. -19: 499, 1980.
3.
Inhibition of antigenBurka, J. F. and N. A. M. Paterson. induced release of prostaglandin-like material from guinea pig trachea by antihistamines, FIX55712 and atropine. J. Pharm. Pharmacol. -32: 869, 1980.
4.
Bawson, W., J. R. Boot, A. F. Cockerill, D. N. R. Mallen, and D. J. Osborne. Release of nnvel prostaglandins and thromboxanes after immunolopical challenge of guinea pig lung. Nature --262: 699, 1976.
5.
Brocklehurst, W. E. Slow-rl>acting substance and related compounds. Progr. Allergy 6: 539, 1962.
6.
Adcock, J. .J. and 1,. G. Garland. A possible role for lipoxygenase products as regulators of airway smooth muscle reactivity. Br. .!. Pharrac. 69: !67, 1980. -
7.
Burka, J. F. and N. A. ?1. Paterson. A comparison of antigeninduced and calcium ionophore A23187-induced contraction of isolated guinea pig trachea. Can. J. Physiol. Pharmacol. 59: in press.
8 ..
Orehek, J., .J. S. Dot~glas, A. J. Lewis, and A. Bouhnys. Prostaglandin regulation of airway smooth muscle tone. Kature New Biol. 245: 84, 1973.
9.
Generation Grodzinska, I,., B. Panczenko, and R. J. Gryglewski. of PGE-like material hy the guink,a pig trachea contracted by histamine.
10.
Constantine, .J.W. The spirally cut tracheal strip preparation. J. Pharm. Pharmacol. 17: 384, 1965.
11.
Ali, M., J. Zamecnik, A.L. Ccrskkls, A. J. Stoessl, W. H. Barnett and .J. W. D. ?IcDonald. Synthesis of thromboxane B2 and prostaglandins by bovine gastric mucosal microsomes. Prostaglandins -14: 819, 1977.
12.
Ali, M. and J. W. II. McDonald. Syntllesis of tllromhoxane B2 and h-keto-prostaglandin F by bovine gastric mucosal and muscle microsomes. Prostaglanc '"i. Ins __* 20. 245, 1980.
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1981 VOL. 22 NO. 5
689
PROSTAGLANDINS 13.
Snedecor, G. W. and W. G. Cochran. Statistical Methods, 6th ed. Iowa State University Press. Ames, Iowa. 1967, I'. 59.
14.
Morrison, D. F. Multivariate Toronto, 1967, p. 117.
15.
Gryglewski, R. J., A. Dembinska-Kiec, L. Grodzinska. and B. Panczenko. In: Lung Cells in Disease. (A. Bouhuys, ed.) Elsevier, Amsterdam, 1976, p. 289.
16.
Boot, J. R., A. F. Cockerill, W. Dawson, D. N. B. Mallen and D. J. Osborne. Modification of prostaglandin and thromboxane release by immunological sensitization and successive immunological challenges from guinea pig lung. Int. Archs. Allergy Appl. Immun. -57: 159, 1978.
17.
Dusting, G. J., S. Moncada and J. R. Vane. Prostacyclin (PGX) is the endogenous metabolite responsible for relaxation of coronary arteries induced by arachidonic acid. Prostaglandins -13: 3, 1977.
18.
Eckenfels, A. and J. R. Vane. Prostaglandins, oxygen tension, and smooth muscle tone. Br. J. Phannac. _* 45. 451, 1972.
19.
Palmer, M. A., P. J. Piper, and J. R. Vane. Release of rabbit aorta contracting substance (RCS) and prostaglandins induced by chemical or mechanical stimulation of guinea pig lungs. Br. J. Pharmac. -49: 226, 1973.
20.
Crews, F. T., Y. Morita, F. Hirata, J. Axelrod, and R. P. Siraganian. Phospholipid methylation affects immunoglobulin E-mediated histamine and arachidonic acid release in rat leukemic basophils. Biochem. Biophys. Res. Commun. -93: 42, 1980.
21.
Engineer, D. M., H. R. Morris, P. J. Piper and P. Sirois. The release of prostaglandins and thromboxanes from guinea pig 64: 211, lung by SRS-A and its inhibition. Br. J. Pharmac. -. 1978.
22.
Diegel, J., M. Cunningham, and R. F. Coburn. Calcium dependence of prostaglandin release from the guinea pig taenia coli. Biochim. Biophys. Acta 619: 482, 1980.
23.
Kaliner, M. and K. F. Austen. A sequence of biochemical events in the antigen-induced release of chemical mediators from sensitized human lung tissue. J. Exp. Med. 138: 1077, 1973.
690
statistical methods.
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McGraw-Hill,
1981 VOL. 22 NO. 5
PROSTAGLANDINS
24.
woreman, J. C., J. L. Mongar, and B. D. Gomperts. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process. Nature 245: 249, 1973.
25.
Kittenhouse-Simmons, S. Differential activation of platelet phospholipases by thrombin and ionophore A23187. J. Biol. Chem. 256: 4153, 1981.
26.
Knapp, H. R., 0. Oelz, L. J. Roberts, B. J. Sweetman, J. A. Oates and P. W. Reed. Ionophores stimulate prostaglandin and thromboxane biosynthesis. Proc. Natl. Acad. Sci. USA -74: 4251, 1977.
27.
Roberts, L. J., R. A. Lewis, J. A. Oates and K. F. Austen. Prostaglandin, thromboxane, and 12-hydroxy-5,8,10,14eicosatetraenoic acid production by ionophore-stimulated rat serosal mast cells. Biochem. Biophys. Acta -575: 185, 1979.
28.
Sun, F. F., .J. P. Chapman, and J. C. McGuire. Metabolism of prostaglandin endoperoxides in animal tissues. Prostaglandins -14: 1055, 1977.
Editor: Priscilla J. Piper Received: 7-28-81 Accepted: 9-23-81
NOVEMBER
1981 VOL. 22 NO. 5
691
IMMUNOLOGICAL AND NON-IMMUNOLOGICAL SYNTHESIS AND RELEASE OF PROSTAGLANDINS AND THROMBOXANES FROM ISOLATED GUINEA PIG TRACHEA. J. F. Burka, M. Ali, J. W. D. McDonald and N. A. M. Paterson Department of Medicine, University of Western Ontario, London, Ontario, Canada. N6A 4G5 ABSTRACT Specific radioimmunoassays were used to demonstrate the synthesis by the guinea pig trachea of 6-keto PGFlu, TxB2, and PGF2o in addition to PGE2. The rank order of both spontaneous and stimulated release was PGE2 > PGF > 6-keto PGFlo = TxB2. Ovalbumin-induced prostanoid release $" rom sensitized tissue was antigenspecific. The release was unlikely to be a secondary consequence of tracheal contraction since incubations with calcium ionophore A23187, at a concentration which produces an equivalent magnitude of contraction of sensitized trachea, did not induce a significant PG or TX production. In contrast, significantly higher prostanoid synthesis was induced by A23187 in unsensitized than sensitized trachea. Thus sensitization altered the profile of arachidonic acid metabolism evoked by the ionophore. Correspondence: J. F. Burka, Department of Pharmacology, University of Alberta, 9-70 Basic Medical Sciences Building, Edmonton, Alberta, Canada. T6G 2H7 INTRODUCTION Activation of arachidonic acid (AA) metabolism occurs following antigen challenge of perfused sensitized guinea pig lung (1) or sensitized tracheal strips (2, 3). The major cyclooxygenase products released immunologically from isolated perfused guinea pig lung are prostaglandin (PG)F2e, prostacyclin (PGI2), thromboxane (Tx)A2 and their metabolites (4). In the guinea pig both large and small airways contract in response to antigen challenge. Since the guinea pig trachea reacts similarly to human bronchus to histamine and slow-reacting substance of anaphylaxis (SRS-A) (5), we have used this model to examine the airways contraction in response to immunological and non-immunological stimuli (2, 3). Indomethacin, a cyclooxygenase inhibitor, reduces airway tone and enhances the contraction induced by antigen, histamine, carbachol (2, 6) and calcium ionophorc 1123187 (7). This may in part be due to inhibition of PG synthesis by the trachea. To date PG release from trachea has been measured only by superfusion bioassay, with which a PGE-like material has been detected (2, 3, 8, 9). We now report the use of specific radioimmunoassays to demonstrate that PGF2o, PG12 and TxA2, in addition to PGE2 are released from the trachea by both immunologic and non-immunologic stimuli.
NOVEMBER
1981 VOL. 22 NO. 5
683
PROSTAGLANDINS
Tracheae were removed from anesthetized male English shorthair guinea pigs (200-300 g) (Connaught Laboratories, Toronto, Ontario), cleaned of extraneous tissue, spirally cut (lo), divided into three equal segments and well washed in Krebs-Henseleit solution. Sensitized tracheae were obtained from guinea pigs immunized 2-4 weeks previously with ovalbumin (DA) 100 mg. S.C. and 100 mg i.p. The first segment was added immediately to 30 ml 07% ethanol. The remaining tracheal tissue was placed in 10 ml oxygenated KrebsHenseleit solution and pre-incubated at 37'C in a gently shaking water bath for 5 minutes. The second segment was incubated for a further 30 min in Krehs solution (i.e. no stimulus added). The third was incubated for 30 min in Krebs solution to which was added AA (100 uM), CIA (10 pg/ml), or A23187 (5.7 yM). The reaction was stop ed by the addition of 2 volumes absolute ethanol, with 0.03 UCi !_ H PGF2 added for recovery analysis. The pH was adjusted to 3 with 88% formic acid and 2 volumes chloroform added. The mixture was well shaken, centrifuged to facilitate phase separation and the chloroform phase evaporated under reduced pressure to approximately 1 ml. The remainder of the solvent was evaporated under N2, the residue resuspended in 0.5 ml 2% methan~\l : 98% chloroform and applied to an activated silicic acid column. 'The column was then eluted successively with 8 ml of 2% methanol in chloroform which removes unused precursor and relatively non-polar products (ll), followed by 8 ml of 10% methanol in chloroform (which contained the more polar prostaglandins and thromhoxanes). The eluate was evaporated under N2 and the residue resuspended in distilled absolute ethanol. Radioimmunoassays of PGE2, PC,F2,,TxB2, and k-keto PGFlcrwere performed as described previously (12). Differences in the amount (rig/gwet weight) of prostnglandin and thromboxane release by trachea in the absence or presence of stimuli were analyzed for significance by the use of Student's t-test (one-tailed) for paired data (13). Comparisons of release from sensitized and unsensitized tissue were analyzed for significance by multivariate analysis using Hotelling's T2 test (2-tailed) (14). OA- and A23187-induced release of prostanoids from sensitized tissue were also compared using Hotelling's T2 test. MATERIALS. Ovalbumin (Grade II for sensitization and Grade III for challenge) and arachidonic acid (Grade I) were purchased from Sigma; calcium ionophore A23187 from Calhiochem; and 3H-PGF2, from Amersham. We are grateful for the gifts of prostaglandins and thromboxane B2 from Upjohn and indomethacin from Merck-Frosst. Indomethacin was dissolved in 1 11Tris buffer (pll8.4), diluted in saline, and kept on ice. AA was stored as a 10 m$;/ml stock solution in hexane under
684
NOVEMBER
1981 VOL. 22 NO. 5
PROSTAGLANDINS
A Tris salt was prcpnred with 1 M Tris buffer (pR 8.4) N’. centration of 1 mg/mi, dilrltcd in sal ine and kept on ice.
to a con-
The chloroform and methanol lrsed in the extraction process were Absolute ethanol was redistilled. toxigrnphic: grade (Annchemia). All other solvents were reagent grade. ~hc silicic acid column was prepared with Bio-Sil A (200-400 A slurry of silicic mesh) (biorad) activated at 115OC for 1 hour. acid in 2L methnnol in chloroform (1:4 wt:vol) was prepared and The column approximately 4 ml added to each column (0.8 x IO cm). in chloroform W;IS waslretl with 6 ml of enc,h of 20% and 2% methanol before application of the snmple. RESULTS. ---~In pilot experiments it was found that maximal release of prostanoids from trachea requirecl 30 min incubation with 10 ug/ml These conditions were OA (in sensitized tissue) or 100 LIM AA. The concentration therefore employed for the remainder of the study. of A.23187 used (5.7 LIM) was that which produced the same magnitude of tracheal contraction as OA (10 pfi/ml) (7). how amounts of h-keto PGFL,r and TxR7L as well as higher amounts of PGE2 and P(:P2o were detected ni~ter immediate extraction of both incubation of the trachea for sensitized and unsensitized trachea. In all situations, 30 min in buffer alone led to additional PG release. including specific stimulatjon witlr AA, CA, or A23187, the rank order of prnstano-itls released was KC2 -- PGF2,> > h-keto P!:FLixg TxR2. iJhen exoj:enous AA (100 tIPI), the natural substrate for cyclowas added to the trachea there was a significant (p d 0.05) oxvgenase increase in the reLease of all four cyclooxygcnase products in both sensitized and unsensitized tracheae.
In sensitized trachea, CIA (10 pg/ml) induced significant increments in tire production of 0-keto PGFLIL, PGE2, and PGF2, (Table The rise in T::B2 approached significance at tile p < 0.05 level Hultivurinte .~nnlysis <.onfirmed a highly significant (p ': 0.07). djfference (p I' 0.001) between the effects of OA on sensitized and In contrast, bovine serum
1).
The calcium ionophore A23187 (5.7 ;IM) failed to stimulate additional PC: or TX relcasc from sensitized trachea (Table 1). In unsensitized tissire nlthou~:l~ the release of the individual prostanoids
NOVEMBER
1981 VOL. 22 NO. 5
685
3
ti
e
4
4
(5.7 PM)
A23187 0
0
(5.7 PM)
OA (10 &ml)
0
0 AA (100 PM)
0
0 0 A23187
87.9 41.3 I 4.6 17.5
14.4 T f 4.8
87.5 54.1 + 5.0 21.4
11.4 T + 1.8
53.2 + 7.3 38.4 + 3.7
27.6 + 2.3 63.3 2 5.5 153.6 + 12.2****
7.6 + 2.0 22.8 + 5.1 99.0 z 21.5* 47.1 f 11.4 47.3 z 7.7
4.6 + 0.7 38.9+ 5.5 34.8 z 0.4
24.0 + 4.0 63.0 + 12.6 172.8 + 21.0***
38.7~+ 4.6 124.6-L 6.5****
4.6 + 0.3
6-keto PGFla
4.1 + 0.6 40.7-+ 16.1 54.0 + 11.4
15.4 + 7.2 101.0+ 8.7 251.9 7 - 75.2
26.0+ 10.1 119.9~+ _ 14.1***
3.2 + 1.3
TxB2a)
462.2 371.6 T + 60.8 110.7
289.9 f 56.4
250.9 t 26.3 334.8 + 53.2
373.3 + 72.9 493.2 + 68.0** 962.4 + 89.3
144.3 + 12.5 251.1 T 33.7 193.1 z 16.6
570.0 + 117.5 899.6 + 79.9 1225.4 -+ 149.4*
208.1 ? 24.8 988.4 z 22.3****
110.6 + 11.4
PGE2
76.3 101.4~+ +- 19.7 30.3
30.2 + 9.7
107.9 + 10.0 86.1 k-4.6
82.7 + 7.1 141.9--t 1.5 398.5 + 25.1**
12.5 + 1.2 46.3 T 5.1 44.9 + 1.9
72.2 •t 7.2 151.8+ 27.0 384.8 T - 20.8****
51.2 + 4.3 211.3+ - 16.4**
16.3 + 0.5
PGF2a
trachea
Statistical significance (* p < 0.05; ** p < 0.025; *** p < 0.01; **** p < 0.005) Values are mean + S.E.M. calculated by Student's t-test for paired data comparing values at 30 min with and without stimulus.
a)
30
2 6 3
4
0 30 30
2
4
0 30 30
.:I? 0
4
4
0 AA (100 UM)
0 0 OA (10 ug/ml)
0
Stimulus
30 30
n
Production of TxB2, 6-keto PGFl,, and PGF2U (rig/g wet weight) by sensitized and unsensitized following stimulation with ovalbumin (OA), AZ.3187 or arachidonic acid (AA).
0
Incubation Time (min)
Table 1.
5; 00
E
5
0
PROSTAGLANDINS
did not achieve significance at the p < 0.05 level, multivariate analysis indicated total prostnnoid synthesis was significantly (p < 0.008) higher than in sensitized trachea.
The present study demonstrates that PGE2 is the major cyclooxygenase product synthesized in guinea pig trachea both spontaneously and following stimulation with M or CA. The pattern is in contrast to guinea pig perfused lung and parenchymal preparations where 6-keto PGF1, and TxB2 are the major AA products (15, 16). This study provides the first evidence that cyclooxygenase products in addition to PGE2 are generated by the guinea pig trachea. Previously, we (2) and others (9) using biological assay methods (i.e. cascade superfusion) detected only a PGE-like substance released by guinea pig trachea in response to CA, AA, ncetylcholine, and histamine, although Orehek et al. (8) reported the release of PGF2c(. Although superfusion bioassay is invalrlahlefor discriminating simultaneously between various M metabolites and for detection of unstable intermediates, the method is limited by its lack of sensitivity. Whereas the threshold for PG12-induced relaxation of the bovine coronary artery is 20-100 ng/ml (17), concentrations as low as 70 pg/ml 6-keto PCFItx (a stable hydrolysis product of PCT2) can be detected by radioimmunoassay. Similar high sensitivities for TxB2, PGE2, and PGF2a are obtainable with radioimmunoassay. Thus it is not surprising that PGI2 and thromboxanes released from the guinea pig trachea were not detected in studies using biological assay methods. The rank order of prostanoids released was PGE2 3 PGF2a > 6-keto PGFle e TxB2. AA normally relaxes the guinea pig trachea (2), an observation which is consistent with the present observation that PGE2 is the major cyclooxygenase product of M metabolism by the trachea. M metabolitcs are not stored to any considerable extent and any increase in their levels induced by a physiological or pathological stimulus occurs through their rapid biosynthesis. Thus it is likely that the basal levels of PGs and Txs detected in these experiments are a consequence of tissue dissection and preparation. Furthermore, the spontaneous generation of PGs and Txs during the 30 min incubation period may have resulted at least partly from membrane pertubations induced by the gentle agitation. Mild mechanical stimulation has previously been shown to induce PG release (19). On the other hand k~ontinuousproductjon of prostanoids in gastrointestinal and tracheal smooth muscle has been reported to contribute to resting tone (8, 18) and the observed spontaneous release may thus reflect physiologic turnover. OA contracts sensitized guinea pig trachea (2) and induces the release of PGE2 and F2d and smaller amounts of 6-keto PGFJ, and TxB2. OA-induced prostanoid release is a consequence of an antigen-specific immunological reaction on sensitized tissue. However it is not known whether M metabolism is activated directly by the immunological
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1981 VOL. 22 NO. 5
687
PROSTAGLANDINS reactions, as has been demonstrated in rat basophilic leukemia cells (20) or secondarily by activation of phospholipase by other released mediators, such as histamine and SRS-A (3, 9, 21). Tt is also possible that the PG release may be secondary to tracheal contraction (8, 9). In the present experiments A23187, employed at a concentration which produces an equivalent magnitude of tracheal contraction (7) did not induce significant PG or TX production. Thus qmthesis of the cyclooqgenase products PGE2, PGF2o, PG12, and TxA2 by trachea is not an inevitable concomitant of smooth muscle contraction. Diegel et al. (22) have suggested that separate calcium pools are involved in smooth muscle contraction (guinea pig taenia coli) and PG synthesis and that the two processes can be dissociated. Whereas 012 initiates an immunological reaction leading to a series of intracellular biochemical events including entry of calcium into the cell (20, 23), calcium ionophore A.23187bypasses the membrane associated events (24) and can activate phospholipase AT by direct introduction of cnlrium into cells (25). A23157 has bc;n reported to stimulate I'(: and TX biosyntllesis in platelets (25, 26) and mast cells (27). It is interesting that significantly higher prostanoid production was induced hy A23167 in unsensitized than sensitized trachea. Sun et al. (28) have suggested that the kinetics of the cyclooxygcnnse differ between sensitized and unsensitized tissue. A fast cyclooxygenase in sensitized tissue would provide lligher amounts of PGR2 which would favour TX synthesis. However, this was not seen in trachea in our experiments and other explanations must be sought. It is possible that cyclooxygenase products other than those measured in our experiments are produced in response to A23187 or that A23187 preferentially stimulates the lipoxygenase pathway of AA metabolism. Both A23187-induced contraction and the prolonged phase of OA-induced contraction are reduced with the cyclooxygenase/ lipoxygenase inhibitor, phenidone, providing indirect evidence that lipoxygenase products contribute to OA- and A23187-induced contractions (2, 7). ACKNOWLEDGENENTS. This project was supported by the Medical Research Council of Canada, the Ontario plinistryof Health, and the Ontario Heart Foundation. We thank Mr. Warren YcDonald and Mr. Chris Webb for their technical assistance, Dr. George Wells for statistical analysis, and Mrs. Sharon Hughes for typing the manuscript.
688
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1981 VOL. 22 NO. 5
PROSTAGLANDINS
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Boot, J. R., A. F. Cockerill, W. Dawson, D. N. B. Mallen and D. J. Osborne. Modification of prostaglandin and thromboxane release by immunological sensitization and successive immunological challenges from guinea pig lung. Int. Archs. Allergy Appl. Immun. -57: 159, 1978.
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Crews, F. T., Y. Morita, F. Hirata, J. Axelrod, and R. P. Siraganian. Phospholipid methylation affects immunoglobulin E-mediated histamine and arachidonic acid release in rat leukemic basophils. Biochem. Biophys. Res. Commun. -93: 42, 1980.
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Editor: Priscilla J. Piper Received: 7-28-81 Accepted: 9-23-81
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